Systems and methods for automated reusable parallel biological reactions

ABSTRACT

A method comprises magnetically holding a bead carrying biological material (e.g., nucleic acid, which may be in the form of DNA fragments or amplified DNA) in a specific location of a substrate, and applying an electric field local to the bead to isolate the biological material or products or byproducts of reactions of the biological material. For example, the bead isolated from other beads having associated biological material. The electric field in various embodiments concentrates reagents for an amplification or sequencing reaction, and/or concentrates and isolates detectable reaction by-products. For example, by isolating nucleic acids around individual beads, the electric field can allow for clonal amplification, as an alternative to emulsion PCR. In other embodiments, the electric field isolates a nanosensor proximate to the bead, to facilitate detection of at least one of local pH change, local conductivity change, local charge concentration change and local heat. The beads may be trapped in the form of an array of localized magnetic field regions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplication Ser. No. 61/389,490, entitled “Integrated System and Methodsfor Polynucleotide Extraction, Amplification and Sequencing,” filed Oct.4, 2010; 61/389,484, entitled “Magnetic Arrays for Emulsion-FreePolynucleotide Amplification and Sequencing,” filed Oct. 4, 2010;61/443,167, entitled “Chamber-Free Gene Electronic SequencingTechnologies,” filed Feb. 15, 2011; and 61/491,081, entitled “Methodsand Systems for Nucleic Acid Sequencing,” filed May 27, 2011, each ofwhich is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under a QualifyingTherapeutic Discovery Grant awarded by the IRS, in conjunction with theDepartment of Health and Human Services. The U.S. government has certainrights in the invention.

BACKGROUND

Methods for quick and cost effective DNA sequencing (e.g., athigh-throughput) remain an important aspect of advancing personalizedmedicine and diagnostic testing. Some known systems for DNA sequencingrequire that DNA samples be transferred between various subsystems(e.g., between the nucleic acid isolation subsystem and theamplification subsystem), thus resulting in inefficiencies and potentialcontamination. Some known methods for DNA sequencing employ opticaldetection, which can be cumbersome, expensive, and can limit throughput.Other systems utilize some forms of electronic sensing, but the sensorand sequencing flow cell are one-time use disposables, whichsubstantially increase the cost to the user, and limits the complexityof the sensor which may be cost effectively manufactured, as it will bethrown out after a single use. Some systems utilize amplificationmethods within the same flow cell, in which the sequencing is performed,binding the amplified directly to the flow cell, preventing reuse. Othersystems utilize emulsion PCR, wherein beads and samples are mixed intosmall emulsions utilizing low concentrations. Due to Poissondistribution, most of the beads and sample do not come together in anemulsion with a single bead and a single sample, and are thus lost. Thecost of the beads is a substantial portion of the cost of thesequencing, and most of that cost is thrown away without ever generatingany useful data. The current system enables utilization of virtually allof the sample, and the reuse of the beads, thus reducing the cost to theuser.

Current DNA sequencing systems typically need whole genome amplificationin order to have sufficient sample, as the sample is very inefficientlyutilized. Such whole genome amplification methods typically introducesignificant amounts of bias in amplification in different portions ofthe genome, and require higher levels of coverage to overcome said bias.Methods for localizing samples, and reagents into a volume wherein adesired reaction or binding may occur is another aspect which isenvisioned for the system, which may eliminate or reduce the need forwhole genome amplification, and thus reduce the coverage needed.

Thus, a need exists for improved systems and methods for extracting,amplifying and sequencing polynucleotides

SUMMARY

The embodiments described herein relate to systems and methods forextracting, amplifying and sequencing polynucleotides. In someembodiments, the systems and methods can include a fully-automated,integrated platform, thereby reducing the cost, improving throughputand/or simplifying the methods of use.

In one aspect, the invention provides a method for isolating biologicalmaterial, reactants, and/or reaction byproducts for a reaction, such asa nucleic acid amplification or sequencing reaction. The methodcomprises magnetically holding a bead carrying biological material(e.g., nucleic acid, which may be in the form of DNA fragments oramplified DNA) in a specific location of a substrate, and applying anelectric field local to the bead to isolate the biological material orproducts or byproducts of reactions of the biological material. Forexample, the bead is isolated from other beads having associatedbiological material. The electric field in various embodimentsconcentrates reagents for an amplification or sequencing reaction,and/or concentrates and isolates detectable reaction by-products. Forexample, by isolating nucleic acids around individual beads, theelectric field can allow for clonal amplification, as an alternative toemulsion PCR. In other embodiments, the electric field isolates ananosensor proximate to the bead, to facilitate detection of at leastone of local pH change, local conductivity change, local chargeconcentration change and local heat. The beads may be trapped in theform of an array of localized magnetic field regions.

In another aspect, the invention provides a method for conductingnucleic acid amplification and/or sequencing. The method comprisesapplying an electric field for confinement of a biological material toan environment, and conducting nucleic acid amplification and/or nucleicacid sequencing on the biological material. The confinement of theenvironment from an external environment via the electric field has theeffect of isolating the biological material into a plurality of regions.The confinement creates a virtual well faciliating amplification and/ordetection, and preventing contamination between virtual wells. Invarious embodiments, the biological material is associated with aplurality of beads, and the beads are held in place by a localizedmagnetic field in each of the plurality of regions. In certainembodiments, amplification within the virtual wells generates a clonalpopulation of DNA associated with each of the beads, or on the surfaceof a sensor.

In another aspect, the invention provides an automated method forseparating a population of beads carrying amplified nucleic acids, froma population of beads not carrying amplified nucleic acids. The methodcomprises separating the populations of beads based on a chargeassociated with the beads. The separation may be implemented withelectrophoresis. The bead separation may be based on a flow-throughmechanism, and the beads may be reused in a subsequent amplificationreaction, for example, by treating the beads so as to remove anyamplified product and/or primer.

In still other aspects, the invention provides a method for purifyingDNA fragments from a biological material. The method comprises applyingan electric field in a fluidic environment, said fluidic environment atleast partially containing a filter medium. In this aspect, the electricfield is adapted to separate a DNA fragment from a biological materialas the biological material is conveyed through the filter medium. Invarious embodiments, the filter medium is a porous membrane or a mediumproviding a different mobility of the DNA fragments compared to aremainder of the biological material. Once purified, the DNA fragmentscan be used for DNA library construction, DNA amplification, DNAenrichment, and/or DNA sequencing, for example, using the methods andsystems described herein.

In yet another aspect, the invention provides a method for shearing DNAisolated from a biological material. The method comprises disposing aplurality of particles in a fluidic environment containing a populationof DNA molecules, and causing flow of the particles in the fluidicenvironment to produce a shearing force on the DNA molecules in order toproduce DNA fragments. In such embodiments, the shearing force producesblunt ends to aid in subsequent library construction.

In another aspect, the invention provides a system for nucleic acidamplification and/or sequencing. The system comprises a substantiallyplanar substrate coupled to a moiety for binding a nucleic acid to thesubstrate, and a means for separating the nucleic acid from thesubstrate such that the system is reusable for at least one of nucleicacid amplification and nucleic acid sequencing. During amplification,the system generates nucleic acid clones on the surface of thesubstrate. Amplification may involve either heating cycles or byisothermal amplification. In various embodiments, the system furthercomprises an instrument for detecting incorporation of a nucleotide in asequencing reaction. The detection may be based on at least one of localpH change, local heat detection, local capacitance change and a localcharge concentration and local conductivity change.

In some aspects, the invention provides a system for detectingbiological material or a biological reaction product or byproduct. Thesystem comprises a substantially planar sensor array, the sensor arraycomprising a means for capturing a bead adjacent to each nanosensor inthe array. The nanosensor is capable of detecting biological material ora biological reaction product or byproduct. The system further comprisesa means for releasing a bead to facilitate reuse of the array, such asby magnetic, chemical, enzymatic means.

In some embodiments of the methods and systems described herein, anapparatus includes a substrate, a porous member and an electrode. Thesubstrate defines a microfluidic channel configured to receive a sample.The porous member is disposed at least partially within the microfluidicchannel. The electrode is configured to produce an electric field, andis coupled to the microfluidic channel such that at least a portion ofthe porous member may be disposed within the electric field. The porousmember and the electric field may be cooperatively configured such thata nucleic acid may be separated from the sample when the sample isconveyed through the porous member.

In some embodiments, an apparatus includes a substrate, a plurality ofparticles and a flow mechanism. The substrate may define a microfluidicchannel configured to receive a sample containing a plurality of DNAmolecules. In other embodiments, the apparatus may be used as a probeand inserted into a well or other fluidic environment. The plurality ofparticles may be configured to be disposed within the microfluidicchannel. The mechanism for producing the flow may be configured toproduce a flow of the sample and the plurality of particles within themicrofluidic channel such that the plurality of particles produces ashear force on the plurality of DNA molecules to produce a plurality ofDNA fragments. In some embodiments, an on-chip peristaltic pump, made ofmultiple fluidic gates with orthogonal control and flow channels (ValveTechnology), or an external pressure may generate the required flow inthe channel.

The present invention provides magnetic arrays and methods of using themagnetic arrays for polynucleotide amplification and sequence analysis,thereby providing fast, convenient, and/or low-cost DNA sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows beads and a magnetic array with sensors which may be usedto capture the beads.

FIG. 1B shows the beads captured on the magnetic array in a one to onecorrespondence with the sensors.

FIG. 1C shows the sensor array after the beads have been washed offready for use for the next sample.

FIG. 1D shows a schematic of a sensor array used with a set ofnonspherical magnetic particles.

FIGS. 2A-2B Show a photomicrographs of a magnetic arrays according to anembodiment loaded with beads

FIGS. 3 and 4 are schematic illustrations of the reaction involved inincorporating nucleotides into a growing DNA strand, showing the releaseof pyrophosphate and concomitant increase in pH and heat release.

FIG. 5A shows a schematic illustration of a series of nanosensors inelectrical communication with the microfluidic channels of thesequencing system.

FIG. 5B depicts a graph showing output of a sequencing operationperformed via a sequencing apparatus.

FIG. 6 shows an array of electronic sensors with a set of electrodesused for concentration or confinement of multiple charged moieties abovethe sensors.

FIG. 7 shows magnetic or electric or electromagnetic retention of aclonal bead held in place for sequencing over a sensor.

FIG. 8A depicts an image of a portion of the sequencing system thatincludes the nanosensors.

FIG. 8B shows a schematic illustration of a nanosensor, and a series ofnanosensors in electrical communication with the microfluidic channelsof the sequencing system.

FIG. 9 shows a schematic illustration of an array of nanosensors withinthe sequencing system. The on-chip amplification is optional.

FIG. 10 depicts components of an exemplary sequencing chip.

FIG. 11 shows the electric forces, a schematic embodiment and aconcentration diagram from a simulation.

FIG. 12 shows the stream line electric field and the electric potentialat a horizontal cross line above the electrodes from a simulation.

FIG. 13 shows a fluorescent micrograph of DNA on beads held by amagnetic array.

FIG. 14 shows a sensor with electrodes for creating electrophoreticconcentration/confinement fields for attracting a sample molecule andconfining the amplification reaction, and its use in an amplificationreaction.

FIG. 15 shows an array of electrical confinement electrodes and sensorsin a one to one correspondence.

FIG. 16A shows a set of beads with clonal DNA attached thereto and asensor and magnetic array.

FIG. 16B shows the set of beads captured wherein each captured DNAcovered bead is held in place over a sensor.

FIG. 16C shows a second set of beads and a sensor and magneticconfinement array which are partially populated with beads.

FIG. 16D shows two sets of beads captured by a sensor and the magneticconfinement array of FIG. 16C.

FIG. 17 shows a sensor with electrodes for creating electrophoreticconcentration/confinement fields used for amplification and sequencingreactions

FIGS. 18A-B illustrate different views of one embodiment of a beadseparation system

FIG. 19 is a schematic illustration of an integrated platform forextracting, amplifying and sequencing polynucleotides according to anembodiment.

FIGS. 20-24 show embodiments of the microfluidic portions of theintegrated platform for extracting, amplifying and sequencingpolynucleotides.

DETAILED DESCRIPTION

As used herein, “bead capture features” may mean features that cantemporarily hold a single bead in a fixed position relative to thesensor and can include local magnetic structures on the substrate,depressions which may utilize an external magnet, local magneticstructures, Van der Waals forces, or gravity as forces that fix theposition of a bead. Optionally, the bead may be bound in place usingcovalent or non-covalent binding.

As used herein, “clonal” may mean that substantially all of thepopulations of a bead or particle are of the same nucleic acid sequence.In some embodiments there may be two populations associated with asingle sample DNA fragment, as would be desired for “mate pairs,”“paired ends”, or other similar methodologies; the populations may bepresent in roughly similar numbers on the bead or particle, and may berandomly distributed over the bead or particle.

As used herein, “confinement” may mean when a molecule generated (suchas DNA) at one bead or particle stays associated with the same bead orparticle so as to substantially maintain the clonal nature of the beadsor particles.

As used herein “isolate” may mean the prevention of migration,diffusion, flow, or other movement, from one virtual well to anothervirtual well as necessary to maintain the clonal nature of the beads orparticles.

As used herein, “localized magnetic feature” may mean a magnetic featurecreated on a substantially planar substrate to hold individual beads onsaid substantially planar substrate.

As used herein, “localized magnetic field” may mean a magnetic fieldthat substantially exists in the volume between the north pole of afirst magnetic region and the south pole of a second magnetic region orsubstantially exists in the volume between the north and south poles ofa single magnetic region.

As used herein, “localized electric field” may mean an electric fieldthat substantially exists in the volume between at least two electrodes.

As used herein, “nanosensor” may mean a sensor designed to detect beadsor particles less than one of 0.1, 1, 5, 10 or 20 micrometers asmeasured on the diameter or the long axis for non spherical beads orparticles. Alternatively, the sensor may be sensitive to moietiesassociated with said beads or particles, or with reaction products orbyproducts wherein the reaction includes a moiety associated with saidbead or particle. Said moieties may include DNA fragments, hydrogenions, or other ions which are counter ions and thus associated with saidbeads or particles or moieties bound or associated with said beads orparticles.

Nanosensors can include “NanoBridge, “NanoNeedle, ISFET, ChemFET,nano-calorimeter or cantilever based pH sensors or combinations thereof.

As used herein, “particle” can mean a non spherical moiety such as amolecule, an aggregation of molecules, molecules bound to a solidparticle, or particles, and other forms known in the art.

As used herein, “single phase liquid” is a liquid with relativelyuniform physical properties throughout, including such properties asdensity, index of refraction, specific gravity, and can include aqueous,miscible aqueous and organic mixtures but does not include non miscibleliquids such as oil and water. Among the physical properties notconsidered to potentially cause a liquid to not be considered a singlephase liquid include local variations in pH, charge density, and ionicconcentration or temperature.

As used herein, “Substantially planar” shall allow small pedestals,raised sections, holes, depressions, or asperity which does not exceed50 μm relative to the local plane of the device. Variations due towarpage, twist, cupping or other planar distortions are not consideredto constitute a portion of the permitted offset. Protrusions ordepressions which are not essential for the uses as described herein butwhich exceed 50 μm do not preclude a device from being consideredsubstantially planar. Fluidic channels and or structures to generatesaid fluidic channels which have dimensions of greater than 50 μm alsodo not preclude a device from being considered substantially planar.

As used herein, “virtual wells” refers to local electric field or localmagnetic field confinement zones where the species or set of species ofinterest, typically DNA or beads, substantially does not migrate intoneighboring “virtual wells” during a period of time necessary for adesired reaction or interaction.

In some embodiments of the current invention, the invention provides anautomated reusable system for performing a sequencing chemistry. In someembodiments a chemistry method performed by the system may includesequencing by synthesis, which is schematically shown in FIG. 3, whereindNTPs bind to a complex which may include a colony of DNA, complementaryprimers, and polymerase. The polymerase incorporates the dNTP to thegrowing extended primer, and creates as byproducts of said incorporationhydrogen ions, pyrophosphate and heat which can be detected byelectronic sensors. By determining whether a base incorporation hasoccurred, or if multiple incorporations occurred, and knowing whatreagents were delivered before such incorporation the sequence of theDNA can be determined

Magnetic Array

The present invention provides magnetic arrays and methods of using themagnetic arrays for polynucleotide amplification and sequence analysis,thereby providing fast, convenient, and/or low-cost DNA sequencing. Themagnetic array may comprise a substrate having a plurality of magneticregions thereon to form the array, the localized magnetic fields beingsufficient for trapping magnetic beads as described herein. Thelocalized magnetic features may be formed from a permanent magneticmaterial (e.g., ferromagnetic), or may be non-permanent and magnetized(and demagnetized) by an electric field.

The array may be formed from any of the known substrate materials, asdescribed, for example, in U.S. Pat. No. 7,682,837, which is herebyincorporated by reference. In certain embodiments, the substratematerial may include at least one of silicon, silicon-based material,glass, modified or functionalized glass, plastic, metal, ceramicplastics or a combination thereof. The substrate is generally anon-magnetic material.

The localized magnetic features may be created with permanent magneticmaterial (e.g., ferromagnetic), or may be non-permanent (e.g.,electromagnetic-induced regions). In certain embodiments, the pluralityof localized magnetic features may be formed from a magnetic material,and each region may be a substantially uniform size and shape, tothereby form an array (e.g., a grid-like pattern), and may thus form aplurality or array of localized magnetic features. In other embodiments,the magnetic features may be non-uniform. In exemplary embodiments, themagnetic features may be magnetic bars, which may be formed at least inpart from a magnetic material comprising, for example, aluminum, cobalt,samarium, neodymium, boron, copper, zirconium, platinum, chromium,manganese, strontium, iron and/or nickel, and including alloys thereof,and may include other materials and elements. In a one embodiment, themagnetic features may be formed at least in part from an alloy of nickeland platinum (e.g., about 50%-50%) or an alloy of cobalt and platinum(80% Co 20% Pt) or an alloy of cobalt, chromium and platinum. Thelocalized magnetic fields may be contained within wells on thesubstrate, or alternatively, the substrate does not contain wells,allowing amplification or sequencing reagents to flow freely over thesubstrate surface, thereby simplifying the sequential addition andcontrol of reagents (e.g., sequential addition of NTPs for sequencing),which can directly improve dephasing and signal to noise ratio for longread sequencing.

In a further embodiment, well structures, depressions, protrusions, orother means of limiting the motion of a bead or particle may be utilizedin combination with localized magnetic fields to retain a bead orparticle in a fixed position, forming a bead capture feature.

Various methods of fabrication may be used for creating the localizedmagnetic features (e.g., magnetic bars). In certain embodiments, thelocalized magnetic features or bars have sharp edges, which may befabricated by photolithography and then sputtering of the magneticlayer. In other embodiments, the localized magnetic features (e.g.,bars) may be fabricated by sputtering/coating of a magnetic layer,followed by photolithography, and then ion-milling to etch off excessmaterial and creating sharp or specific angle edges. In someembodiments, the fabrication may utilize multi-layer resist lithography.

The localized magnetic features may be configured to be in a singledomain state. The localized magnetic features may be fabricated with anumber of layers, alternating between a ferromagnetic material and anintermediate layer of another material such as chromium, in order toimprove the coercivity of the multilayer magnetic structure. In additionto the alternating layers, there may be a seed layer and a protectivelayer of a material such as Tantalum, MgO or other appropriate materialsas known in the art. There may be a number of alternating layers, forinstance 2 to 40 layers, for example, 2 to 4 layers, 5 to 10 layers, 10to 16 layers, or 16 to 30 layers, or 32 layers or more. The grainorientation may be parallel to the long axis on the localized magneticfeatures. The thickness of the layers may vary from 5 nm to 15 nm ormore for each layer.

In some embodiments, the localized magnetic features may be rectangularprisms, with a length of about 20 microns, with a width of one to 2microns, and gaps for holding a bead or particle may be 2 to 3.5 micronswhen the diameter of the bead is 4.5 microns. The lengths, widths, andgaps may all be scaled up or down as appropriate for a larger or smallerbead or particle. For example, for a 2.8 micron bead, the localizedmagnetic features may have a length of 10 microns, a width of 1 to 2microns, and a gap for holding said bead or particle of from 1.25 to 2.5microns.

The array may be a high density or low density array. The arraysgenerally contain at least 100 magnetic regions per mm², and in certainembodiments, contain at least 1,000 localized magnetic features per mm²,and in certain embodiments contain at least 100,000 localized magneticfeatures per mm². The array may contain at least 1,000, 2,000, 4,000,5,000, 10,000, 100,000, 1,000,000, 10,000,000 or 100,000,000 or morelocalized magnetic features.

The localized magnetic fields may be sufficient for trapping (bymagnetic force) magnetic particles having a size of 50 μm or less. Incertain embodiments, the localized magnetic fields may be sufficient fortrapping magnetic particles having a size of 20 μm or less, 5 μm orless, 500 nm or less, or 50 nm or less. In certain embodiments, thebeads have a diameter of from about 3 μm to about 5 μm, and in otherembodiments the beads have a diameter from about 0.5 μm to 3 μm. Themagnetic particles may be ferromagnetic, paramagnetic, orsuperparamagnetic, and suitable materials are well known, as describedin U.S. Pat. No. 7,682,837. The beads may be moved into the array byflow, for example, via a channel having a height of from about 5 to 50μm, such as about 15 μm. The width of the channel may vary, but in someembodiments may be from about 500 μm to 1 mm, such as about 800 μm. Inother embodiments the channel width may be from 1 mm to 20 mm or more.In some embodiments the channel may have supporting posts or ribs tobetter control the height. In other embodiments, parallel channels maybe utilized, either to accommodate more array positions for a singlesample, or to accommodate multiple samples.

In some embodiments of the current invention, wherein magnetic beads orparticles are utilized without a magnetic array, said magnetic beads mayself assemble into a monolayer with uniform spacing. In otherembodiments the self assembling beads or particles may be nonmagnetic.In some embodiments depressions associated with the sensors canfacilitate a one to one correspondence and may result in betteralignment between the beads and the sensors permitting better detection.Slow translation or movement of the beads may be appropriate afterconditions have been caused to be appropriate for binding, in order toenable alignment of the beads with the sensors. Such translation ormovement may occur in multiple dimensions, which may include x, y,theta, and may have varying movements in time and distance toaccommodate the spacing of the sensors and the size of the beads. Inother embodiments, a circular fluidic movement may be used to ensurehigh rates of bead loading.

In designs with deep wells beads or particles may not be adequatelyaccessible to fluid flow. In some embodiments, the beads or particlesare more accessible to fluid flow, as they may protrude above thesurface of the device. As a result, the beads may respond more quicklyto introduction of reagents, permitting better, quicker and moreefficient washes and reactions.

FIG. 1A schematically illustrates the addition of beads 102 to amagnetic array 104A. FIG. 1B schematically illustrates the positioningof said beads in a one to one correspondence with the retention regionson the array 104B. FIG. 1C shows the sensor array after the beads havebeen washed off ready for use for the next sample.

FIG. 1D illustrates various embodiments of the current invention whereinthe magnetic, paramagnetic, non magnetic particles or a combinationthereof may be of shapes other than spherical for use with either asensor array 104C with magnetic retention, a sensor array withelectrical confinement not shown or a sensor array with self assembledparticles 104D. In one embodiment said particles may be substantiallyplanar. The substantially planar particles may be round, rectangular106A, star shaped, hexagonal 106B, or in another shape. In otherembodiments, the particle may be dendritic including a dendriticstructure formed by a self assembled 3D DNA network, enlarging thesurface area of said particle. Said dendritic particle may be generallyspherical, substantially planar, oval, or any other shape. In someembodiments, primers may be attached said dendritic particles. In otherembodiments DNA nanoballs may be attached to dendritic particles orother types of particles or beads. In yet other embodiments, saidparticle may be porous or partially porous; if said particle is porousor partially porous, the pore size may be of sufficient size as topermit free movement of DNA, polymerase, dNTPs and other moietiesnecessary for primer extension sequencing or other applications asappropriate In all places where the term bead is utilized, it may beassumed that it may be of any shape as described herein.

FIGS. 2A and 2B are micrographs of localized magnetic arrays filled withmagnetic beads, as described in various embodiments herein, illustratingthe routinely high occupancy level achievable and illustrating a furtherembodiment of the current invention, wherein a single magnetic orparamagnetic bead may be held in place in a single position in themagnetic array. Said beads may be sized such that there may besufficient room for only one bead over each sensor, thus providing for aone to one correspondence between array positions and beads. Althoughthere may be room for only one bead over each sensor, there can be anadditional distance between beads when the beads may be aligned inproximity to the sensors, resulting in reduced cross-talk betweensensors. For example, a set of beads may be 10 microns in diameterlocated over sensors which may be 8 microns across, and the sensors maybe spaced 15 microns apart, resulting in a 5 micron space between thebeads. The size of the sensors may be larger than the beads, if there isinsufficient room for two beads to be retained above the sensor. Thesize of the beads, sensors, and spacing can vary. In other embodiments,beads may be greater in size than 10 microns, such as 15 microns, 20microns, 25 microns, or larger. In further embodiments the beads may besmaller than 10 microns, such as 5 microns, 3 microns, 2 microns, 1micron, or less than one micron. The sensors may be sized to align withthe size of the beads, and thus may be larger, or smaller in size than 8microns across, potentially ranging from less than one micron, to 1, 2,3, 5, 10, 15, 20 or more microns across. The spacing between the sensorsmay also be greater than 15 microns, or may be less than 15 microns; thesensor spacing may range from less than one micron, to 1, 2, 3, 5, 10,15, 20, 25 or more microns between sensors. The sensors can be arrangedin a square, rectangular, hexagonal or other 2-D pattern. Althoughdescribed herein primarily for DNA applications, includingamplification, real-time PCR, sequencing, digital PCR, DNA hybridizationprobe array, the magnetic arrays may be utilized for other applications,such as applications or methods utilizing and or detecting antibodies orother proteins.

In some embodiments with 4.5 um diameter magnetic beads a flow rate of0.07-0.14 mm/sec may be desirable for bead loading to allow capture bythe localized magnetic fields. A flow rate of 1.4-4.2 mm/sec may bedesirable for reagent delivery to prevent dislodging of the magneticbeads. A flow rate of >5.6 mm/sec may be desirable for bead removal. Inother embodiments the use of air bubbles can be used to remove thebeads. Larger and smaller beads may be used with higher and lower flowrates although the relationship may not be linear.

Other Reuse Methods

After a set of sequencing cycles has been completed, the primers may beremoved and replaced. Buffer conditions can be changed to weaken abiotin streptavidin bond, such as high concentrations of GuHCl at lowpH; alternatively the temperature can be raised to over 70 C to breakthe biotin streptavidin bond. Lower temperatures may also be used withlow ion strength buffers, such as buffers with micro molar saltconcentrations. Combinations of the above may also be utilized, such ashigher temperatures and low ionic strength buffers. Thiol bonds canlikewise be broken at elevated temperatures. Aggressive means may beutilized, as damage to the polymerase and DNA may be no longerconsequential, as the sequencing reaction has already been completed. Inone embodiment, organic reagents may be utilized to break the bindingbetween the extended primer and the surface, such as a covalent binding.After the extended primers have been removed, new primers may be flowedinto the volume above the sensors, enabling the device to be used againfor another set of sequencing cycles on another set of DNA samples. Saidnew primers may be bound in a single phase liquid. Said new primers mayalso have additional reagents included in the fluid containing saidprimers which assist binding or associating of the primers to thesensors. The new primers may be utilized in an amplification reaction togenerate a new clonal population for subsequent sequence analysis, asdescribed herein. Said amplification may be PCR or isothermalamplification.

In a further embodiment, an amplification or sequencing array may bereused by the removal of beads. Said removal may be done, for example,by the application of an external magnet field, which may result fromthe movement of a permanent magnet or the activation of an electromagnet, to pull, move or dislodge beads or particles from wherein theyare held in said amplification or sequencing array.

In an alternative embodiment, wherein said beads or particles are heldin place with a Biotin Streptavidin binding, thiol binding, DNA, LNA,PNA, or other nucleic analog hybridization, or the like, the release ofsaid binding may be achieved by changing the temperature and or fluidicenvironment proximate the bead or particle, so as to reversibly breakthe binding, so that new beads or particles may be subsequently bound orassociated in the amplification or sequencing array.

Sequencing

FIGS. 3 and 4 are schematic illustrations of the reaction involved inincorporating nucleotides into a growing DNA strand, showing the releaseof pyrophosphate and concomitant increase in pH. As described herein,the integrated sequencing platform may include an electronic sensingsubsystem configured to electronically detect the change in pH or chargeconcentration or mobility to “electrically sequence” the DNA. In otherembodiments, an electronic sensing subsystem can be configured toelectronically detect the change in temperature resulting from thisreaction to “electrically sequence” the DNA.

FIG. 5A depicts two sets of beads, one with clonal sets of DNA bound orattached thereto, and a set without clonal sets of DNA bound or attachedthereto. This system permits utilization of the beads without clonal DNAbound or associated thereto to be used as a reference, removing offset,nucleotide and other reagent charge, temperature, fluidic flow andpressure, buffer concentration changes and other systematic variables tobe removed. As shown in FIG. 5A, in schematic system 510, said removalof system variables may be done at least in hardware, using an analogsubtraction. In other embodiments, the removal of systematic variablesmay be performed is in software and or external hardware. In yet otherembodiments, a combination of local hardware and software and orexternal hardware may be utilized. FIG. 5B depicts resultant data,wherein most putative in corporation reactions result in signal levelsindicative that a reaction did not occur, while some putativeincorporation reactions result in signal levels indicative of a singleincorporation event, and other putative incorporation reactions resultin signal levels indicative of multiple incorporation events in ahomopolymer region of the clonal DNA.

In a further embodiment, an electronic sensing subsystem may detect achange in conductivity, either in a bulk solution, across the surface ofthe sensor (either from moieties bound to the sensor or from moietieswithin the Debye length of the surface of the sensor), across thesurface of a bead or particle (either from moieties bound to the bead orparticle or from moieties within the Debye length of the surface of thebead or particle), or a combination thereof. In a yet furtherembodiment, an electronic sensing subsystem may detect a change incharge near or on the surface of the sensor, near or on the surface of abead or particle. For example, the electronic sensing subsystem maydetect charge changes within the Debye length of the surface of thesensor, or bead or particle, or of moieties bound to the surface or beador particle.

FIG. 6 includes a schematic illustration of a nanosensor 604 and aseries of nanosensors 610 associated with the microfluidic channels inelectrical communication with the sequencing system. The nanosensors mayhave clonal DNA 602 bound or associated directly thereto, and may haveelectrodes or magnetic elements 608 associated with each nanosensor. Inother embodiments the sensor may detect changes in the charge of theclonal DNA on the bead, changes in the counter ions associated with saidclonal DNA, or byproducts which result from an incorporation. Thenanosensor 604 may further include a signal amplifier 606 for on-chipsignal amplification. The nanosensors 604 may further include any of theknown insulator materials, such as SiO₂, Al₂0³, SiN, and TaO₂In certainembodiments, the nanosensors may comprise coaxial and/or parallelconductive layers, separated by an insulator layer. The conductivelayers may be formed from any suitable material, such as gold, platinum,aluminum, carbon, or polysilicon.

The (magnetic) beads and DNA fragments may be conveyed into thesequencing system. As shown in FIG. 7, the sequencing system may includea series of nanosensors 708 in communication with the microfluidicchannels defined within the sequencing system. The beads or particles702 may be positioned over said sensors 708 by magnetic or electrodeelements 710, which may form localized magnetic fields in someembodiments and may form localized electric fields in other embodiments,wherein both the sensors 708 and magnetic elements may be configured inassociation with a substrate 712. The beads or particles 702 may haveclonal DNA 704 bound or associated thereto. Reagents, which may includenucleotides, primers, magnesium and polymerase 706 may then be providedto initiate a sequencing reaction. In other embodiments, when magneticor electrode elements 710 are magnetic elements, they may be eitherpermanent magnetic elements or electromagnetic elements.

In other aspects, the invention provides a method for sequencing apolynucleotide, using a magnetic array, forming an array of localizedmagnetic features as described herein. The method comprises contactingthe magnetic array with a plurality of magnetic beads, the magneticbeads each having attached thereto a clonally amplified DNA segment,which may be single stranded, partially double stranded or doublestranded. Whether single stranded, partially double stranded, or doublestranded, the template DNA may be converted to single-stranded DNA bydenaturation and a sequencing primer may be hybridized to thesingle-stranded DNA to prepare for sequencing.

After base-calling, the recorded sequence at each location on the arraymay be assembled. For example, by using a shot-gun sequencing method,wherein the identities of the fragments at each position of the arraymay be unknown, or a polynucleotide sequence may be assembled based upona reference sequence (e.g., a wild-type sequence).

The clonal DNA sequences may each have a single-stranded region, actingas a template for nucleotide incorporation. The single stranded regionmay be at least 10 bases in length, or in some embodiments, may be atleast 300 bases in length, or in other embodiments, at least 1 kb inlength. The invention thereby provides for long, accurate, and costeffective reads. There may be more than one amplified populations ofpolynucleotides in one clonal population as defined herein, wherein thedifferent amplified populations of polynucleotides may have differentprimers, so that separate sequencing reactions may be performed for eachof the amplified populations within a single clonal population.

In another aspect, the magnetic array comprises an adjacent nanosensorfor determining a change in pH of a microenvironment, themicroenvironment including the environment in the vicinity of the beadheld by the localized magnetic field. In this aspect, the microarray maybe useful for electronic sequencing of DNA. Methods for sequencing bydetecting a change in pH are generally described in U.S. PatentPublication No. 2008/0166727, which is hereby incorporated by referencein its entirety. Alternative methods of detecting incorporation ofpolynucleotides may be used, including thermal sequencing (e.g., asdescribed in U.S. Patent Publication No. 2008/0166727), detection ofcharge concentration, mobility of charged species and byproducts, andknown optical detection methods.

The magnetic array comprises a substrate having a plurality of localizedmagnetic features thereon to form the array, the localized magneticfields being sufficient for trapping magnetic beads as described herein.The localized magnetic features may be formed from a permanent magneticmaterial (e.g., ferromagnetic), or may be nonpermanent and magnetized(and demagnetized) by an electric field.

In other embodiment, an electric field may be used to hold or retain abead or particle in a location as will be described later herein.

Detector

A magnetic or paramagnetic bead or particle may be held in place over orproximate a sensing region by a magnetic array, forming an array oflocalized magnetic fields. Retained magnetic or paramagnetic beads mayhave monoclonal populations of DNA. Said beads may be sized such thatthere may be sufficient room for only one bead over each sensor, thusproviding for a one to one correspondence between sensors and beads.Although there may be room for only one bead over each sensor, there canbe an additional distance between beads when the beads may be alignedover the sensors, resulting in reduced cross-talk between sensors.

The magnetic sequencing array comprises a plurality of nanosensors, withat least one or two nanosensors in the vicinity (microenvironment) ofeach of the localized magnetic fields. The nanosensors have a highsensitivity for detecting slight changes in pH or charge concentrationin each microenvironment (e.g., the vicinity of each localized magneticfield). For example, an array may comprise 1000 nanosensors or more,2000 nanosensors or more, 4000 nanosensors or more, 10,000 nanosensorsor more, 100,000 nanosensors or more, 1,000,000 nanosensors or more.10,000,000 nanosensors or more or 100,000,000 nanosensors or more. Thenanosensors may comprise measuring electrodes having two terminals,sufficient to determine an increase in the ionic (H⁺) concentration, oran increase in the counter ions associated with DNA in the correspondingmicroenvironment or the occurrence of the polymerization reaction.

The nanosensors may include at least one pair of measuring electrodeshaving positive and negative terminals, sufficiently spaced apart (e.g.,a spacing of between 20 and 30 nm) and constructed to detect a change inthe ionic concentration of the corresponding microenvironment. In otherembodiments the spacing between the electrodes can be 100 nm to 500 nmor 1000 nm to 5000 nm. More particularly, the nanosensor can detect achange in the impedance of the fluid within the microenvironment causedby a change in the ionic concentration of the correspondingmicroenvironment as a result on an incorporation event or a chemicalreaction of the biological material on the beads and another material.In an alternative embodiment, the sensor can be a resistivesemiconductor element as described in U.S. Provisional patentApplication No. 61/389,590 entitled “Biosensor Devices, Systems andMethods Therefore.” In yet another embodiment, the nanosensor may be aChemFET or ISFET, as described in U.S. Pat. No. 7,695,907 “Genedetection field-effect device and method of analyzing gene polymorphismtherewith”, U.S. Pat. No. 7,948,015 entitled “Methods and Apparatus forMeasuring Analytes Using Large Scale FET Arrays,” U.S. PatentApplication No. 2011/0171655 entitled “pH Measurement for Sequencing ofDNA” and U.S. patent application Ser. No. 13/118,044 entitled“Nano-Sensor Array,” each of which is hereby incorporated by referencein its entirety. Whenever the term nanosensor is utilized herein, it maybe considered to be a set of electrodes as described above, or may be aresistive semiconductor element, or may be an ISFET or ChemFET orcombination of the abovementioned sensors.

In some embodiments of the current invention, a combination of differentsensing methods may be utilized, for example, a NanoNeedle and aNanoBridge, or an ISFET and a NanoNeedle. In some embodiments, thedifferent sensors may sense different properties associated with thetarget moieties. For example, a NanoNeedle may detect the conductivityof moieties bound or associated with the target moieties, while aNanoBridge may detect charge bound or associated with the targetmoieties.

FIG. 8A shows a photomicrograph of an array of nanosensors, and a zoomedin view of a single nanosensor 802. Impedance measurements may be usedby such a nanosensor for detecting incorporated nucleotides. Theimpedance measurement detects the release of H+ ion pyrophospate orlocal change in charge resulting from the polymerization reaction.Generally, the frequency of operation may be selected for maximum changein the impedance over the course of the reaction relative to theimpedance at the beginning of the reaction. For example, for somegeometries, the frequency may be around 0.1 to 9 KHz. In alternativegeometries, the frequency may be 10 KHz or greater. In some embodiments,the nanosensor may be implemented with a single pair of electrodes withor without a pH-sensitive material (e.g., redox sensitive material) todetect the H+ ion release or pH change of the reaction. The impedancemeasurement may be taken, as an example, by determining the currentwhile sweeping from −A to +A volt or the reverse, with periodicsub-signals. A pulse wave with smaller amplitude than A, and a frequencyof about 25 Hz or above, can be applied. A measurement of the currentduring a voltage sweep may indicate a change of pH in the solutionproximate the nanosensor. FIG. 8B shows a schematic illustration of anarray of said nanosensors 802, wherein an on chip amplifier 804 may beassociated with each nanosensor.

FIG. 9 is a schematic illustration of an array of nanosensors 902 withinthe sequencing system. The nanosensor may comprise two electrodes 904,separated by a dielectric 906. Although shown in FIG. 9 as including anarray of nanosensors, in other embodiments, the measurement can be donewith a single electrode pair to detect the change of ionic constructionor pH through impedance, charge, or current measurement.

The system may be calibrated for sequence analysis as follows. To reducethe common noise and signals from various environmental sources (e.g.,thermal noise, mixing or fluidic noise, or the effect of nucleotidecharges or other reagents), one or a plurality of beads(s) without DNAmay be located in a similar environment as the DNA-coated beads. Adifferential measurement between the recorded signals from the twosensors (detecting the microenvironment of a DNA-coated-bead andbead-without-DNA or sensor without bead) dramatically reduces the noise,and results in an improved signal-to-noise ratio during detection. Insome embodiments multiple local reference sensors can be combined tocreate a local average reference. In other embodiments magnetic featurescan be left off creating sensor positions with no beads. In someembodiments, the differential measurement may be done by comparing aneighboring DNA bead with no reaction in a cycle with the bead ofinterest for the same cycle. In other embodiments, the neighboring beadsfor differential measurement may be chosen from the region that receivethe fluidic flow at substantially the same time, or beads without DNA orwith DNA and without a reaction in that cycle. In other embodiments,averaging of the background signal over more than a single cycle may beused. Differential measurement of the sensor with another sensor whichis shielded from contact or interaction with the fluid or targetmoieties.

In some embodiments, the integrated sequencing platform can produce abetter signal to noise ratio, reduce the noise level from the proton (H+ion) and OH− effect in sequencing detection and/or produce betterisolation in virtual wells, than may be currently possible using knownsystems and methods. More particularly, in some embodiments, systems andmethods can employ a buffer media configured to improve the performance,as stated above. The buffer can have different mobility and diffusioncoefficients for H+(proton) ions than the coefficients would be inwater. The buffer can also have different changes in the coefficientsfor H+ and OH−. In some embodiments, a buffer media can be a materialvery similar to water, but with different mobility of H+, such asDeuterium oxide (D₂O or heavy water) or any common material having thisfunctionality. The difference in mobility can slow the movement of H+ion released in polymerization reaction. In another aspect, the buffermedia can include material having different mobility for H+ ions and/ordifferent materials e.g. DNA, nucleotides, primers or other moieties,and can be a gel-type material. A gel-type material would result indifferent mobility and diffusion for H+ ions released within thegel-type material, and facilitates easier detection, resulting to abetter signal to noise ratio.

To calibrate the system for sequencing, and to ensure that the recordedsignals from individual sensors may be appropriate and correct, a commonsequence of nucleotides may be embedded in all template DNA strandsbeing sequenced. This common sequence may be introduced during theamplification stage by design of the amplification primer. For example,a sequence of AATCGA may be incorporated at the front end of allsequences, and may be utilized to calibrate the system, allowing knownreadouts of each of the nucleotide incorporations, also permittingcalibration of a single base incorporation as opposed to a two or morebase incorporation. Any combination of bases could be utilized, whichcould utilize all four of the bases, three of the bases, two of thebases, or a single base, and could include single base incorporations,two base incorporation, or any number of bases, up to and includingeight base incorporations or more. Different primers may also be used asa means for encoding different samples.

Electrical Confinement and Retention

In one embodiment, a magnetic array may comprise electrodes positionedto create an electric field around each of the localized magneticfields, to thereby concentrate template DNA, polynucleotides and dNTPsaround the localized magnetic fields (e.g., by electroosmostic,electrophoretic or dielectrophoresis force) to thereby enhance apolynucleotide amplification or polymerization reaction. The electricfields can create isolation between the regions of the array during thePCR or sequencing process, conduct DNA strands and/or nucleotides orother charged molecules toward the beads for clonal PCR, and/or conductnucleotides toward the DNA-coated beads for sequencing. For example,electrodes may be positioned under the bead capture positions and inseveral positions surrounding the bead capture regions, such as in acircular or square arrangement, so as to enhance the polymerizationreaction. The magnetic array for sequencing analysis may be created on anon-magnetic substrate as described. The read-out circuitry and on-chipamplifiers, which may be in pixelated structure, may be implementedabove the substrate. Subsequently, the individual nanosensors may befabricated, which may be in contact, directly or indirectly, with themicroenvironment of the reaction as shown in FIG. 10. The magnetic bararray 1006 generates localized magnetic fields to associate the beads inthe proximity of the sensors 1008. Optional associated amplifiers 1004may be fabricated above or below the sensor layer as shown in FIG. 10 aspart of an integrated device 1002. Microfluidic channels may be embeddedin the structure. The chip may be operably connected with a dataacquisition unit. In other embodiments, bead retention in bead capturefeatures may occur utilizing a localized electrical field. In someembodiment the bead or particles can be nonmagnetic. Yet furtherembodiments may comprise electrodes positioned to create an electricfield around each of the bead capture feature, sensors or other desiredlocations, to thereby concentrate template DNA, polynucleotides anddNTPs (e.g., by electroosmostic, electrophoretic or dielectrophoresisforce) to thereby enhance a polynucleotide amplification orpolymerization reaction. The electric fields can create isolationbetween the regions of the array during the PCR or sequencing process,conduct DNA strands and/or nucleotides or other charged molecules towardthe beads for clonal PCR, and/or conduct nucleotides toward theDNA-coated beads for sequencing.

FIG. 11 schematically illustrates some of the forces which combine tolocalize the charged moieties with lower diffusion constants in adesired volume, including the electrophoretic flow which may result froman impressed electric field, frictional force, electrostatic force, andelectrophoretic force. The schematic 1108 shows a voltage source 1118generating a voltage impressed on the electrodes 1106, to generate alocalized electric field.

The localized electric field may comprise AC and or DC components, andmay utilize non-sinusoidal waveforms. Said non-sinusoidal waveforms maycomprise triangle waves, square waves, or waves of any shape. Saidnon-sinusoidal waveforms may comprise a “dead spot” in, for example thepeak of a sinusoidal waveform, in order to allow hybridization binding,enzymatic binding, other binding, and enzymatic activities to occurwithout the presence of a potentially interfering electric field. Other“dead spots” could be utilized for example, in a square wave, whereinthe voltage could be raised to level of A volts for a period of time,and then be reduced to zero volts for a period of time. The voltagecould then be raised to A volts again, followed by an amplitude ofnegative A volts. The “dead spot” need not be zero volts, but can bereduced sufficiently so that a desired interaction between differentmoieties influenced by the electric field may occur. The result oflocalized electric field on the charged molecule concentration 1109shows the substantial gradient which results from the electric field andmay provide substantial isolation.

Although described herein primarily for DNA applications, electricalconfinement as described above may be utilized for other applications,such as applications or methods utilizing and or detecting antibodies orother proteins or chemical metabolites. In some embodiments, otherreactions other than sequencing or amplification may be performed in aset of virtual wells. For practical usage in such an application, themoieties which need to be isolated need to be charged or associated withother charged moieties.

FIG. 12 shows electric potential at a horizontal cross line 1209 abovethe electrodes from a simulation 1208 which results from an electricalfield being applied to the electrodes 1206. The stream line electricfield 1202 and electrical potential due to the DC voltage which may beused for capturing charged moieties, including DNA amplicons near beadsand preventing them from migrating toward the next bead. This simulationwas performed for dNTP migration.

Amplification

In one embodiment of the current invention, the magnetic bar andelectrode array provides for an emulsion-free method of clonallyamplifying DNA fragments on magnetic beads, by isolating regions of thearray by magnetic and or electric fields. Clonal amplification on beadshas been generally described in U.S. Pat. No. 7,323,305, which is herebyincorporated by reference in its entirety. The invention may employbridge amplification, which immobilizes the DNA strands on a surface ofa bead, particle or sensor during amplification, thereby furtherpreventing diffusion of DNA strands to other beads, particles, orsensors.

In an exemplary method for amplifying DNA fragments, magnetic beads maybe injected onto the magnetic bar array having electrodes forming anelectric field. DNA strand templates (double-stranded or singlestranded) may be injected into the chamber to go over the beads in aconcentration targeted for a desired DNA-strand per bead distribution,thereby allowing for clonal amplification. In certain embodiments, toinsure that polyclonal regions are not generated, the concentration ofinput DNA needs to be low enough that most sensor regions have one orzero sample DNA molecules. dNTPs and DNA polymerase may then be injectedinto the chamber, and may be concentrated around the beads by virtue ofan electric field as described. DNA primers for amplification may beprovided at any step, such as when adding dNTPs and/or polymerase, orprovided with the DNA templates. The DNA fragments immobilized on thebeads may be amplified by PCR or isothermal amplification. Where doublestranded DNA is the starting material, the first step of theamplification process creates single-stranded templates by “melting” thedouble stranded fragments, followed by primer annealing and extensionsteps, and repeated heating cooling cycles if PCR is utilized, or by acontinuous controlled temperature for an isothermal amplification. FIG.13 shows a fluorescent photomicrograph of clonal beads with doublestranded DNA held in an array as described herein.

During the amplification process, dielectrophoresis forces may also aidin preventing cross contamination between different sensor regionsundergoing amplification by retaining amplicons. In the embodimentillustrated in FIG. 15 the additional electrodes are shown as having thesame voltage relative to voltage level of the sensors. In an alternativeembodiment as shown in FIG. 14 electrodes on either side of a sensor mayhave voltages of opposite sign or the same sign with different valuesrelative to each other.

In addition, a gel-type material can act as an isolating material in andor between different regions during amplification or sequencing with amagnetic array. The use of such a gel-type buffer media can result inminimal diffusion of DNA strands from one localized magnetic field tothe neighbor (or adjacent) localized magnetic field, because thenucleotides (dNTPs), Mg2+ and other materials may be introduced duringcyclic injection and can be transported through the gel-type or spongymedia. The gel-type material can be any suitable material, such asagarose or acrylamide or other cross linking materials, in which crosslinking may be initiated through physical or chemical triggers. Oneexample of such triggers is a change of temperature (as a physicaltrigger), or the addition of a substance (to produce a chemical changeto the material to make the media into the gel-type phase).

A “gel-like” or “spongy” material can also help confine the DNA strandsin the volume near the beads, or help confine the DNA strands in or nearthe localized magnetic fields and/or reduce the diffusion of thepolynucleotides. In such embodiments, the nucleotides and othermaterials may be allowed to diffuse more readily, but DNA strands,particularly sample or amplicon fragments may be impeded from freelydiffusing.

In some embodiments, this method may reduce the diffusion of the DNA inthe amplification portion of the system.

FIG. 14 depicts an alternative embodiment, wherein a clonal populationmay be generated in the area, or on individual sensors 1402 in a sensorarray. The sensors may be NanoNeedles or NanoBridges or other sensors todetect the event of polymerization. In one embodiment, primers 1404 maybe preferentially bound, associated with or attached to the surface ofthe sensors. Said primers 1404 may be preferentially attached as aresult of a difference in materials, wherein the material of the sensormay be more advantageous for attachment then the areas between thesensors of the sensor array. In an alternative embodiment, a mask may beapplied to areas between the sensors of the sensor array, and a surfacemodification may then be performed. Subsequently, the mask may beremoved; leaving an area between the sensors of the sensor array whereinthe surface modification has not been performed. The surfacemodification may include attachment of biotin, applying a layer of goldand various other methods as are known in the art.

Primers 1404 may then be preferentially applied to the areas on thesurfaces of the sensors 1402 in the sensor array. In one embodiment, theprimers could be attached as a result of a biotin streptavidin binding,wherein the biotin or streptavidin may be attached to the 5′ end of theprimers. In another embodiment, a thiol group may be attached to the 5′end of the primers, which can then bind to the gold layer previouslyapplied above the sensor, forming an Au—S bond. If a PCR reaction isdesired, the primers may be modified with DTPA such that two thiol goldbonds may be formed, preventing the dissolution which may otherwiseoccur from the 60 to 95C temperatures routinely used in PCR. Target DNA1406 may be concentrated in the area of the primers 1404 by electricfields 1406 generated by electrodes 1408A and 1408B. Primers, dNTPs1410, and polymerase 1408 may be introduced and optionally concentratedby electric fields generated by electrodes 1408A and 1408B.

In some embodiments, amplification may be a solid phase amplification,wherein one primer may be on the surface of the bead, and a secondprimer may be in solution. In other embodiments, the amplification maybe solid phase wherein all primers are on the bead, or the amplificationmay be performed whereby both primers are present in solution, and oneprimer or both primers may be also present on the bead. In a furtherembodiment, the amplification may be performed whereby one primer ispresent in solution, and one primer or both primers are also present onthe bead.

The electrode configuration may take various different forms, includinga substantially planar electrode on one or both major planes of the flowcell, or there may be an electrode on the surface opposite the beads,and a set of smaller electrodes associated with each sensor, or beadcapture region FIG. 15 schematically illustrates one embodiment, whereina set of sensors 1502 may be located associated with electrodestructures 1504 and 1506, wherein the electrode structure may have anelectrode 1504 located in immediate proximity to the bead capturefeature, and a larger electrode structure 1506 may encircle the beadcapture feature and the smaller electrode 1504. The bead capture featuremay be in proximity to the sensor active area when the embodiment usedfor sequencing or detection. The larger electrode is illustrated as acircle, but it may me a square, a rectangle, an oval or other shape, andneed not completely encircle the smaller electrode 1504.

The substantially planar structure may include depressions or wells forbetter alignment or field focusing, or pedestals for better fluid flowcharacteristics.

Prior to amplification the beads may be associated, in some embodiments,with a single DNA fragment in order to create monoclonal beads.Typically the DNA concentration may be determined and then the DNA maybe introduced to beads in a dilute form so that on average less than 1fragment may bind to each bead. Many beads have zero DNA fragments,fewer have a single fragment and a small number have 2 or morefragments. The steps needed for quantitation often require a separateinstrument and separate processing. Frequently quantitation may be doneutilizing real time PCR, or determination of the absorption at 260 nm.

If three or more electrodes are utilized, different voltages may beutilized for any set of the electrodes.

A polymer may be utilized in conjunction with an AC field which has onephase with a higher voltage and shorter duration in order to providedirected mobility of the target molecules.

In one embodiment the sample could be made very dilute and/or a smallvolume of sample reagent may be utilized and loaded onto beads. DNAwould bind to some of the beads and then be amplified in the virtualwells creating beads with DNA. The sequencing primer could be madeshorter than the complement ligated to the sample DNA. Since thesequence is known, the correct dNTPs could be added and detected. In oneembodiment multiple dNPTs could be simultaneously added. For example, ifall dNTPs are added the polymerase would extend to the end of thefragment generating a large signal. Said large signal could be generatedas a part of the amplification process. This would allow the detectionand counting of the number of beads that have DNA even if the beads hadminimal amplification. Knowing how many beads have signal would allowcalculation of the proper dilution to generate the ideal number ofmonoclonal beads. The signal may be electrical, optical or any othertype of the detection signal known in then art.

In some embodiments, electric confinement of amplicons, polymerase orother moieties may be utilized with a device which does not have anyphysical well structure. In other embodiments, the device may be asubstantially planar surface, wherein depressions or protrusions exist.In yet other embodiments, the device may have well structures.

Electrical Concentration

As illustrated in FIGS. 6 and 12, the sensor array may be provided withan additional array of electrodes, which may be utilized to performdielectrophoretic concentration. Dielectrophoretic concentration may beinitially performed to attract sample DNA 1206 dNTPs 1210, primers, andpolymerase 1208, to each sensor or amplification region, permittinglower concentrations of each said moiety to be utilized. Amplificationcan then commence in the region of each sensor where a sample DNAmolecule may be located. The electric field generated virtual well canprevent amplicons from leaving one virtual well and traveling to anothervirtual well, generating cross contamination. In a similar manner, thefields used to localize the amplicons may also concentrate theamplicons, primers, and polymerase to the region of the sensor oramplification region.

In another embodiment the sample may be concentrated in theamplification region using the existing electrodes of the emulsion freenano-well. In one embodiment electrodes may be established on a singleplane. In another embodiment electrodes may be added to a second planeparallel to the plane of the virtual wells. In other embodimentsmixtures of AC and or DC voltage inputs are anticipated.

In another embodiment dielectrophoresis could be used to concentrateDNA. During or after concentration the electrical current could bemeasured to determine the DNA concentration. In another embodiment theconcentrated DNA could be quantitated by the use of intercalating dyesas described below.

The isolating field electrodes may also be utilized for concentration.In some embodiments the same electrodes and field may be utilized. Inother embodiments, fewer or more electrodes may be utilized to generatethe concentration field, relative to those used for generating anisolating field.

Concentration may be utilized to maximize utilization of sample, forexample, directing or pulling DNA sample to virtual wells for subsequentamplification. Concentration may also be utilized to direct or pullpolymerase to a virtual well for amplification, or to a clonal set ofDNA which may be associated with a bead or sensor, and said polymerasemay be utilized in a sequencing reaction. In a similar manner, othermoieties such as dNTPs, primers, other enzymes, and other biological orother charged moieties may be concentrated for use in a reaction, or usein a subsequent reaction.

Significant amplification of sample DNA is often performed to ensuresufficient DNA sample is available at a high enough concentration forthe desired protocol. This amplification can introduce bias and may bean additional cost in time and resources. The ability to reduce oreliminate the need to amplify the sample may be desirable. In oneembodiment the beads to be loaded may be enclosed in a packed bed andsample may be pumped across it. In some embodiments the sample can bepumped through the area with the beads multiple times to provideadditional opportunities for the sample to bind. The high surface areato volume should allow minimal sample to be used. The beads cansubsequently be moved into a flow cell whereby they may be held in placeby a magnetic array, and local colonies may be created on the beads byPCR or isothermal amplification.

Multiple Samples

Since many projects do not require the full use of the chip it may bedesirable to load multiple samples in a single chip. In one embodiment,samples may be directed into separate zones separated by walls on thechip using valves integrated into the chip assembly. Such valves couldbe polydimethylsiloxane PDMS valves integrated into the fluidic path. Inanother embodiment, samples may be directed into separate zonesseparated by walls on the chip using valves separated from the chipassembly with multiple inputs on the chip assembly. In anotherembodiment there may be separate zones with separate inputs and outputs.In another embodiment samples may be directed into separate zones on achip or flow cell using a local electric field. A positive field may beapplied to attract DNA or DNA-coated beads to desired regions, while anegative field may be applied to repel DNA or DNA-coated beads fromundesired regions. In another embodiment samples may be directed intoseparate zones using electromagnets to separate magnetic or paramagneticbeads. In another embodiment samples can be delivered into individuallanes using self sealing ports. Self sealing ports can include rubbersepta and needles.

In another embodiment samples can be injected at different time pointsand new beads can be distinguished due to signal from previously emptybead locations.

In some embodiments of the current invention, as a part of the samplepreparation process, “barcodes” may be associated with each sample. Inthis process, short oligos are added to primers, wherein each differentsample utilizes a different oligo in addition to primer. The primers andbarcodes are ligated to each sample as part of the library generationprocess. Thus during the amplification process associated withgenerating each colony, the primer and the short oligo are alsoamplified. As the association of the barcode is done as part of thelibrary preparation process, it is possible to utilize more than onelibrary, and thus more than one sample, in generating the clonalpopulations, permitting determination of which bead and colonyoriginates with which sample, by sequencing the short oligo along withthe sample sequence.

Sample separation methods can be used in conjunction with sampleidentifiers. For example a chip could have 4 separate channels and use 4different barcodes to allow the simultaneous running of 16 differentsamples. This permits the use of shorter barcodes while still providingunambiguous sample identification.

In an alternative embodiment as shown in FIGS. 16 A-D, samples may bebrought into a system which may have a magnetic array and associatedsensor array. Alternatively the system may have a combined amplificationand detection array, wherein each element of the array may have a sensorand a set of electrodes configured to create a virtual well. A DNAsample set 1604A which is configured to occupy only a portion of saidarray 1602A, may be introduced to said array 1602A, resulting in aportion of the available areas to have an associate sample. Such samplesmay then be detected by the sensors associated with each virtual well,resulting in an array 1602B as shown in FIG. 16B, or may be amplifiedand then detected. FIG. 16B also shows a photomicrograph of a partiallyfilled magnetic array. FIG. 16C shows a further sample set 1604B, whichmay then be introduced to the magnetic and sensor array 1602B, resultingin a more completely filled array 1602C as shown in FIG. 16D.

Combined Electrical Confinement and Sequencing

FIG. 17 illustrates the use of the amplified regions above the sensorsin the array of sensors which may be used in a sequencing reaction. DNAsample 1702 may be brought into the array system 1710, wherein the arraymay be configured either with pre-localized beads, or with primers 1708which may be attached, bound or associated with sensor regions 1710.Polymerase 1704, dNTPs 1706 and additional primers may besimultaneously, previously, or subsequently introduced to the array.After the amplification reaction 1712 has been completed, the volumeabove the sensor array may be washed, removing amplicons, polymerases,and dNTPs, resulting in locally bound associated or attached clonal setsbeing associate with array positions. Polymerase 1718, primers 1714, andindividual dNTPs 1706 may then be flowed into the volume above thesensor array, permitting binding, incorporation, and detection of theincorporation events, resulting in the determination of the sequence ofthe different amplified sample DNA molecules. Polymerase 1718 used forthe sequencing reaction, may be the same type of polymerase 1704 as usedfor the amplification reaction, or may be a different type ofpolymerase.

Separation of Clonal Beads

Part of FIG. 19 shows a schematic illustration of a system which mayseparate magnetic or paramagnetic beads with clonal DNA from magnetic orparamagnetic beads which have not had amplification product associatedthereto and/or have incomplete amplification and/or short clonal. Themagnetic beads 1934 may then be separated such that magnetic orparamagnetic beads 1934 having a clonally amplified DNA segment boundthereto may be conveyed into the sequencing system, and magnetic orparamagnetic beads 1934 that are largely devoid of amplified DNA may beconveyed to a waste chamber and/or retained within the PCR andenrichment system. The separation or “enrichment” may be produced byapplying an electric field across a portion of the PCR and enrichmentsystem to induce electrophoretic flow. Thus, the magnetic orparamagnetic beads 1934 having amplified DNA, which is highly charged,may be efficiently separated from those magnetic paramagnetic beads 1934largely devoid of amplified DNA. In this manner, the sample delivered tothe sequencing system can include substantially only those beads havingamplified DNA with a desired length of DNA strands for sequencing.Similarly stated, the sample delivered to the sequencing system caninclude a percentage of clonal beads approaching 100%. The separation ofclonal beads may be non-magnetic beads or any other type of the beads,with or without the surface being coated with charged molecules.

When generating clonal beads a large percentage of the beads may have noDNA template. In addition others may have poor amplification. Thesebeads do not provide useful sequencing so it may be desirable to removethese beads for better efficiency. In some embodiments of the currentinvention an enrichment module may be used which separates the beadswith no or minimal amounts of template using an electric field.

Beads fully loaded with templates have a higher charge, and so may movefarther in an electric field than beads with only primers or fewtemplates. In one embodiment as shown in FIG. 18A-B this separation maybe done in a flow through module 1800. A first fluidic input 1811Aallows the injection of mixed beads. A second inlet 1812A allows theinjection of a buffer solution without beads. A first outlet 1811B maybe downstream from the first inlet 1811A. A second outlet 1812B may bedownstream from the second inlet 1812A. Fluids may be brought into orout of the module through ports 1809. The fluidic system may have asubstrate 1802, and a channel 1810 formed in a layer of PDMS 1808 glassor other material.

The fluidic flow rates can be set by fluidic resistance or pumping speedsuch that more liquid flows in the second inlet. In one embodiment theinlet and outlet widths may be varied to create different fluidicresistances but other methods of modifying the fluidic resistance suchas different length, height are anticipated. Similarly the fluidicresistance of the first outlet 1811B and second outlet can be modifiedso more liquid flows out the first outlet 1811B. In such a setup beadswithout a small velocity perpendicular to the flow may exit the firstoutlet port 1811B. Additional output channels can be added to facilitateseparation of beads with medium levels of template.

A pair of electrodes 1813 may be provided which enable generation of anelectric field perpendicular to the fluid flow such that the templateloaded beads migrate out of the flow path towards second outlet 1812B.Fluidic ports 1809 allow connection to the system plumbing.

The electrodes could be made of any electrode material compatible withelectrophoresis. In some embodiments discrete metal wires may be usedbut metal traces are also anticipated. Metals such as platinum,platinum/iridium, gold and other noble metals or alloys are anticipatedas well as corrosion resistant materials such as stainless steel. Nonmetal electrodes such as carbon are also anticipated.

The flow through enrichment module chamber can be constructed of nonconducting materials such as molded plastic, glass, ceramic or moldablepolymers such as PDMS. Fluidic components can be fused or bonded tocreate a flow chamber.

The voltage applied to the electrodes can be reduced or even reversedperiodically if necessary should beads stick to the electrodes. Thevoltages used should be greater than that required for electrolysis(1.23V at 25 C at pH 7). Higher voltages and narrower gaps provide ahigher field strength and more force on the beads. The voltage on thesystem can be calibrated by flowing beads without or with limitedtemplate and adjusting the voltage or flow rate such that these beadsmay not be moved far enough to enter the second outlet while beads withtemplate may be directed into the second outlet.

Non flow-through enrichment modules are also anticipated but these maynot be as easily automated as flow through systems. In one embodimentbeads may be introduced to a chamber and a magnetic field or gravitypull the beads down. An electric field may be established pulling thebeads with template up. In some embodiments a capture membrane or filtercan be added in front of the positive electrode to facilitateconcentration of the beads.

In some embodiments, beads or particles which do not have amplified DNA(clonal beads), and or beads or particles which have insufficientlyamplified DNA, or beads and or beads or particles which have amplifiedDNA fragments which are too short, may be recycled and reused for asubsequent amplification reaction in order to generate well amplifiedclonal beads or particles. Beads or particles may also be recycled aftersaid beads or particles have been utilized for sequencing. Said beads orparticles may be recycled automatically within a single system.

In some embodiments, beads or particles which do not have amplified DNAmay be directly reused or recycled without further processing of thebeads or particles to prevent contamination from sample to sample. Thismay be advantageous, for example, when a single sample is utilized forseveral amplification reactions, rendering any cross contaminationirrelevant, as the sample is in fact the same. In other embodiments, theamount of cross contamination which may result may be consideredinconsequential, as the amount of cross contamination is sufficientlylow.

In other embodiments, the beads may be treated to prevent crosscontamination. Said treatment may, for example, comprise removal andreplacement of all primers from said beads or particles wherein saidprimers may be associated or bound to the beads or particles using, forexample, streptavidin binding, thiol binding, or the like, wherein thebinding may be broken and another moiety bound. The primer which isbound to the beads or particles may be the same primer as was previouslyutilized, or may be a different primer, having for example, a differentbarcode included as part of the primer.

In other embodiments, cross contamination may be prevented by utilizingprimers with an unusual nick site, wherein the primer may be nicked,washed, a splint oligo provided and the primer restored by ligation ofan oligo wherein the original sequence, or another desired sequence forthe oligo is regenerated or generated.

Integrated System

FIG. 19 is a schematic illustration of the integrated sequencingplatform. The integrated sequencing platform may include a DNAextraction system, a library construction system, an amplificationsystem an enrichment system, and a sequencing system (which may includethe electrical detection system or “sensing unit” described herein).Although shown schematically as separate systems, the integratedsequencing platform can include all of these systems within a singlemicrofluidic/microelectronic device (or “chip”). Each of the systems isdescribed in more detail below.

The DNA extraction system includes an inlet chamber 1910 for receivingthe biological sample (e.g. blood) to be analyzed. The inlet chamber caninclude a solution to promote lysing of the cells contained within thebiological sample. Such solutions are well known in the art and aretypically called lysis buffers. In some embodiments, the lysis solutioncan be injected into the inlet chamber and mixed with the biologicalsample. The DNA may be extracted from the biological sample via anon-chip extraction element 1920. The extraction element 1920 can bedisposed within a flow channel of the microfluidic device, and includesa filter media constructed from a porous member. The extraction element1920 may also include one or more electrodes configured to produce anelectrical field across the filter media. Thus, the combination of thefilter media and the electrical field causes separation of the highlycharged DNA (identified by reference character DNA) from the otherportions of the biological sample. Moreover, the extraction element 1920can be configured to separate DNA 1912 from other nucleic acids (i.e.,RNA).

In some embodiments, the electrodes can be controlled to tailor thecharacteristics of the electric field, thus optimizing the separationcharacteristics of the extraction element. For example, the electrodescan be controlled to adjust the strength, polarity, spatial variabilityand/or transient characteristics of the electric field. In someembodiments, the extraction element 1920 can include two electrodes: thefirst being disposed under the porous filter media, and the second beingdisposed above and diagonally from the first.

As shown in FIG. 19, the library construction system may include a DNAfragmentation and/or size selection element 1916. The fragmentationand/or size selection element 1916 can be configured to producedouble-stranded DNA fragments having blunted ends via the elements andmethods described below. The fragmentation element 1920 includes one ormore microfluidic channels 1922 within which the separated DNA may bedisposed along with a set of fragmentation beads 1924. Moreparticularly, the separated DNA produced by the DNA extraction systemcan be conveyed or “injected” into the DNA fragmentation and/or sizeselection element 1916 by any suitable mechanism (e.g., pressurizedinjection, electrophoretic movement, gravity feed, heat-inducedmovement, ultrasonic movement and/or the like). Similarly, thefragmentation beads 1924 can be conveyed into the DNA fragmentationand/or size selection element 1916 by any suitable mechanism.

The fragmentation and/or size selection element 1916 may include a pump1926 to produce movement of the solution of DNA and fragmentation beads1924 within the microfluidic channel 1922. The pump 1926 can be, forexample, a peristaltic pump. In some embodiments, the pump 1926 caninclude one or more microfluidic elements in fluid communication withthe microfluidic channel 1922, and having a flexible side-wall that,when deformed, produces a flow within the microfluidic channel 1922. Inother embodiments, however, any suitable mechanism can be used toproduce movement of the solution of DNA and fragmentation beads 1924within the microfluidic channel 1922 (e.g., via selective heating andcooling of the solution, pneumatic pressurization of the microfluidicchannel, electrophoretic motion, or the like.)

The fragmentation beads 1924 can be constructed from any materialsuitable for separating, cutting and/or otherwise dividing the DNA intoDNA fragments (identified by reference character DNA-SEG). In someembodiments, the fragmentation beads 1924 can be constructed from glass,polydimethylsiloxane (PDMS), ceramic or the like. Moreover, thefragmentation beads 1924 can have any suitable size and/or geometry suchthat the fragmentation element 1920 produces DNA fragments having thedesired characteristics (e.g., length, strand characteristics or thelike). Moreover, the size and/or geometry of the microfluidic channel1922 (e.g., cross-sectional shape, aspect ratio or the like) can beselected such that the movement of the DNA within the microfluidicchannel 1922 and in contact with the fragmentation beads 1924 producesthe desired shearing of the DNA. For example, in some embodiments, thefragmentation beads 1924 can be substantially spherical and can have adiameter of 50 μm or less. In other embodiments, the fragmentation beads1924 can have a diameter of 500 nm or less, or any diameter between 50μm and 500 nm. In some embodiments, the microfluidic channel 1922 may bein the range of 1 to 500 μm in hydraulic diameter (i.e., as shown inFIG. 24, the cross-sectional area of the microfluidic channel 1922 canbe substantially rectangular, thus the size can be represented as ahydraulic diameter). In other embodiments, the hydraulic diameter of themicrofluidic channel 1922 can be in the range of 10 to 200 μm. In yetother embodiments, the hydraulic diameter of the microfluidic channel1922 can be in the range of 500 nm or less. Moreover, although shown inFIG. 24 as being substantially rectangular, in other embodiments themicrofluidic channel can have any suitable shape, such as semi-circular,oval, tapered or the like. In some embodiments enzymatic polishing ofthe sheared DNA ends can be done to insure the ends are blunt ends.

In other embodiments, an enzymatic solution can be conveyed into themicrofluidic channel 1922 to, at least partially, produce enzymaticfragmentation of the DNA.

Upon completion of the fragmentation, the DNA fragments may be separatedfrom the fragmentation beads 1924. The DNA fragments can be separatedfrom the fragmentation beads 1924 by any suitable mechanism, such as,for example, by a filter, by gravitational (or centripetal) separation,by an electrical field, or the like. In some embodiments, for example,the DNA fragments can be separated from the fragmentation beads 1924 bythe actuation of one or more control lines or control channels, asdescribed below with reference to FIG. 20. In particular, the controlchannels may be channels that are fluidically isolated from, butadjacent and usually perpendicular to the microfluidic channel 1922. Thecontrol channels can, for example, be defined by a side wall that alsodefines a portion of the microfluidic channel. In this manner, when thepressure of a fluid within the control channel may be increased, thecommon side wall can deform, thereby changing the flow area of a portionof the microfluidic channel 1922. To separate the DNA fragments from thefragmentation beads 1924, a pressure can be selectively applied to thecontrol channel such that the flow area of the microfluidic channel maybe small enough to retain the fragmentation beads, but large enough toallow the DNA fragments to pass therethrough. Said another way, in someembodiments, the valves in the channel can be partially closed creatinga leaky “sieve valve” to separate the DNA fragments from thefragmentation beads 1924.

In some embodiments, the fragmentation and/or size selection element maycomprise an electrophoretic device which may further comprise a set ofelectrodes embedded in a microfluidic channel and may further include ameans for introducing an entangled polymer, buffers and wash solutions.

As further shown in FIG. 19, the DNA fragments may then be conveyed intothe amplification and enrichment systems. The amplification andenrichment systems can be configured to produce clonally amplified DNAfrom the fragmented DNA that can be sequenced as described below. ThePCR and enrichment system may include an array of microfluidic channels1932 within which the DNA fragments may be associated with a series ofmagnetic beads 1934. The DNA fragments and magnetic or paramagneticbeads 1934 may be positioned within the microfluidic channels via acorresponding magnetic array. In this manner, the DNA fragments andmagnetic or paramagnetic beads 1934 can be maintained in the desiredposition to promote accurate and efficient sample amplification. Forexample, the DNA fragments and magnetic or paramagnetic beads 1934 canbe maintained in the desired position within the “flow-through”microfluidic channels 1932, and the desired reagents can be conveyedwithin the microfluidic channels 1932 and into contact with the DNAfragments to promote amplification of the DNA fragments.

After amplification of the target DNA onto beads, the beads may besorted in an electrophoretic sorter 1938 as previously described, andbeads with appropriate amounts of amplified product 1940 may be movedinto a sequencing module 1936.

As described above, the integrated sequencing platform can include allof the systems described herein within a singlemicrofluidic/microelectronic device (or “chip”) or may be modulardevices in one system. FIGS. 20-24 show embodiments of the microfluidicportions of the integrated platform for extracting, amplifying andsequencing polynucleotides.

As in FIG. 20, a microfluidic device 2000 may have one or more input oroutput ports 2006. Fluids may be introduced through said ports 2006 tofluidic channels 2004. Control lines 2002 may control the flow of fluidsthrough the activation of valves 2008. Pressurizing the control lines2002, deforms a wall between the control lines 2002 and the fluidicchannels 2004, pinching off the fluidic channel and closing the valve2008. FIG. 21 shows a further embodiment of the fluidics system, withsimilar control lines 2102, fluidic channels 2104 and valves 2108, butwith additional crossovers 2110, wherein the control line is narrowedtoo much to be able to fully deform to the point wherein the fluidicchannel 2102 is sealed, preventing the crossover 2110 from acting as avalve 2108. FIG. 22 shows microphotographs of a portion of a device2200A and 2200 B with enlarged fluidic channels 2204, permittingvisualization of the activation of control line 2202. If the view of thedevice 2200A, the control line is not activated, and the valve 2208 canbe seen to be open. After activation of the control line 2202, one cansee that in device 2200B that the valve 2208B has deformed and sealedthe fluidic channel 2204. FIG. 23 shows several views of a PDMS valvingdevice 2302, including photomicrographs of paramagnetic beads 2304 in achannel, and view with a wherein flow is occurring, and where a valvehas been activated and no flow occurs. The microfluidic device cancontrol the “injection” or flow of the beads, reagents and/or samplesdescribed herein by a series of control lines that intersect with and/orimpede upon the microfluidic channels described herein. As shown in FIG.24 and described above, the control lines can be expanded to retain thesolution and/or beads within a predetermined portion of the device.

For injecting picoliter amounts of amplification or sequencing reagentsinto the fluidic system, e.g., for incorporation of dNTPs ontobead-immobilized DNA primers, the magnetic array may utilize amicro-fluidics system. For example, the microfluidic platform maycontain lines for injecting/delivering reactants to the localizedmagnetic fields. For sequencing embodiments, the microfluidic systemcontrols sequential injections of nucleotide triphosphates to thesubstrate or to localized magnetic fields. The microfluidic channels maybe in the range of 1 to 100 μm in diameter, or in certain embodiments,in the range of 10 to 20 μm in diameter. Materials and methods forfabricating the micro-fluidics system are known. For example, themicrofluidics system may be fabricated with polydimethyl siloxane(PDMS), molded or machined plastic or glass.

The invention therefore provides in certain aspects, a magnetic array,as described herein, having a magnetic bead or particle trapped bymagnetic force at a plurality of the localized magnetic features, eachmagnetic bead or particle having bound thereto a clonally amplified DNAsegment for sequence analysis. The DNA segment may be clonallyamplified, for example, using the magnetic array, optionally having anelectric field, as described herein.

The amplification and sequencing arrays may be placed in sequentialorder in an integrated platform. For example, after amplification on amagnetic array, the beads may be enriched based on a DNA electrophoreticforce. Specifically, the beads with amplified DNA, and with the adequatelength, may have the minimum required charge to be pulled off to an exitintegrated with a DNA sequencer. The null beads, as well as beads withincomplete amplification or overly short DNA amplicons, may be separatedthrough another outlet.

It may be desirable to process multiple samples in a single chip, sincemany projects do not require the full capacity of a chip. Other projectsmay have a single sample that would exceed the capacity of the chip. Insome embodiments one or more samples could be introduced into theinstrument in individual tubes, tube strips, 96-well plates, 384-wellplates, etc. In some embodiments the sample wells could be sealed toprolong life on the instrument. In other embodiments the plates may becooled to increase sample life. In other embodiments the samples couldbe accessed in a software selectable manner by a robotic pipettor. Thesystem could divide the samples over multiple fluidic channels or chipsif they are too large, or combine the samples if they are combinable(for example using sequence barcoded samples). In some embodimentssample may be loaded at different times in the same sequencing device indifferent channels, enabling samples to be run when they becomeavailable. In some embodiments samples provided to the instrument wouldbe ready for sequencing. In other embodiments samples could be processedby the instrument to generate sequencing ready samples.

In one embodiment a target concentration may be created by ahybridization based pullout. A solid support such as pull-out beadscould be functionalized with a controlled number of binding sites. Insome embodiments these could be DNA primer compliments. The unamplifiedsample may have known primers ligated on each end. In some embodimentsthe primers would hybridize to the DNA on the pull-out beads. After thesites are exhausted, residual DNA would be washed away, and the DNAbound to the beads would subsequently be denatured releasing a knownquantity of DNA.

In another embodiment the primers ligated to each DNA fragment could bebound to the primer compliment and detected using fluorescence detectionof an intercalating dye. Since the primers may be of a known length, thesignal level may be proportional to the number of fragments. In anotherembodiment polymerase and associated dNTPs could be introduced creatingfull length double stranded DNA. When combined with the information fromthe primer signal the full length intercalating dye signal level wouldthen allow determination of the mean fragment length.

Although various embodiments have been described as having particularfeatures and/or combinations of components, other embodiments arepossible having a combination of any features and/or components from anyof embodiments as discussed above.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods and/or schematics described above indicatecertain events and/or flow patterns occurring in certain order, theordering of certain events and/or flow patterns may be modified. Whilethe embodiments have been particularly shown and described, it will beunderstood that various changes in form and details may be made. Whilethe embodiments have been particularly shown and described for nucleicacid detection or DNA sequencing, it will be understood that the systemmay be configured or used for various other biochemical reactions anddetection thereof.

1.-14. (canceled)
 15. A device for nucleic acid sequencing, comprising: a surface configured to immobilize a plurality of nucleic acid molecules in solution during sequencing; a flow cell in fluid communication with said surface, wherein said flow cell is configured to direct a plurality of nucleotides to said plurality of nucleic acid molecules during said sequencing; and a sensor array in fluid communication with said flow cell, wherein said sensor array comprises a plurality of sensors, wherein a sensor of said plurality of sensors comprises at least two electrodes adjacent to said surface, which at least two electrodes are configured to contact said solution during said sequencing, wherein said at least two electrodes comprise a first electrode oriented along a first plane and a second electrode oriented along a second plane that is different than said first plane, wherein said at least two electrodes are configured to detect impedance, charge, or conductivity, or change in impedance, charge, or conductivity, of said solution adjacent to said surface during said sequencing, which impedance, charge, or conductivity, or change in impedance, charge, or conductivity, is indicative of incorporation of a nucleotide of said plurality of nucleotides into a nucleic acid molecule of said plurality of nucleic acid molecules.
 16. The device of claim 15, wherein said surface and said sensor array are part of a chip.
 17. The device of claim 15, wherein said surface is part of a planar array.
 18. The device of claim 15, wherein said surface is part of a well.
 19. The device of claim 15, wherein said surface is configured to immobilize a particle comprising said plurality of nucleic acid molecules.
 20. The device of claim 19, further comprising a magnetic array configured to immobilize said particle adjacent to said sensor.
 21. The device of claim 20, wherein said magnetic array comprises a plurality of magnets, and wherein a magnet of said plurality of magnets is disposed adjacent to said sensor.
 22. The device of claim 21, wherein said magnet comprises a permanent magnet.
 23. The device of claim 21, wherein said magnet comprises a non-permanent magnet provided by an electric field.
 24. The device of claim 15, wherein said sensor array does not comprise wells.
 25. The device of claim 15, wherein said first electrode and said second electrode are separated by a dielectric.
 26. The device of claim 15, further comprising circuitry configured to (i) receive signals from said at least two electrodes and (ii) use said signals to determine said impedance, charge, or conductivity, or change in impedance, charge, or conductivity.
 27. The device of claim 26, wherein said circuitry is configured to, based at least in part on said impedance, charge, or conductivity, or change in impedance, charge, or conductivity, identify said nucleotide of said plurality of nucleotides.
 28. The device of claim 26, wherein said circuitry comprises read-out circuitry, on-chip amplifiers, or both read-out circuitry and on-chip amplifiers.
 29. The device of claim 15, wherein said first plane is oriented above said second plane.
 30. The device of claim 15, wherein said nucleic acid molecule is part of a clonal population of nucleic acid molecules, and wherein said clonal population of nucleic acid molecules is disposed adjacent to said sensor.
 31. The device of claim 15, wherein said sensor array is configured to reduce cross-talk between said sensor and another sensor of said plurality of sensors. 